siRNA / RNAi /miRNA transfection Human Cells THP-1

Get tips on using Absolutely RNA miRNA Kit to perform RNA isolation / purification Cells - immortalized HT-29

Get tips on using HyClone Dulbecco's Modified Eagle Medium (DMEM)/ F12 1:1: Liquid to perform 3D Cell Culture Media hiPSC-derived cortical organoids

Get tips on using HyClone Dulbecco's Modified Eagle Medium (DMEM)/ F12 1:1: Liquid to perform 3D Cell Culture Media hiPSC-derived forebrain organoids

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Tissue - Human Salivary glands

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Tissue - Human Bone marrow

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.

Get tips on using pPG-1-FlaA to perform Protein Expression Prokaryotic cells - E. coli surface flagellin A

ROS has a very short half-lives in biological environment as they are influenced by exposure to ambient oxygen. As it is highly reactive and hard to measure care should be taken to ensure the stability of the sample during isolation, preparation, storage, and analysis.

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - immortalized A-172